The major objective of Project by Bolton continues to be to provide in vitro bioassay support for the discovery of the active components for the major dietary supplements for the relief of menopausal symptoms. To achieve this objective several in vitro cell based and animal assays have been developed in order to investigate the mechanism of action of these dietary supplements. Specifically, we are interested in botanicals with estrogenic activity such as hops (Humulus lupulus), kudzu (Pueraria lobata), and kwao keur (Pueraria mirifica). In addition, we have discovered an alternative serotonergic mechanism for the relief of menopausal symptoms which could be important for black cohosh (Cimicifuga racemosa), dong quai (Angelica sinensis), and valerian (Valeriana officinalis). The hypothesis to be addressed is whether select plant extracts contain potent estrogenic and/or serotonergic compounds useful for the relief of menopausal symptoms as well as other beneficial properties of estrogens such as antioxidant/chemopreventive effects. The following specific aims are proposed: 1. To evaluate the hormonal activity of plant extracts and active compounds. We have already screened a number of plant extracts and pure compounds for estrogenic effects and investigated their mechanism of action using a variety of in vitro assays including competitive ER binding assays, induction of estrogen sensitive genes, and ERE-luciferase assays. In addition, we have tested several of these extracts for hormonal activity in vivo using the ovariectomized rat model. We have also incorporated two new screening assays including the estrogen receptor coactivator assay and an assay to measure ERE and AP-1 DNA binding capacity. 2. To evaluate the serotonergic activity of plant extracts and active compounds. We plan to further fractionate black cohosh (aim 3) in order to determine the active compounds as well as test other promising plants using established serotonergic assays (competitive binding to serotonin receptors, elevation in cyclic AMP activity, and SSRI assays), addition, we plan to further test the hypothesis that the 5-HT7 receptor is involved in thermoregulation (i.e. hot flashes) whereas the 5-HT1A and 5-HT5A receptors are likely responsible for depression and mood. Animal models will be incorporated including the hot flash model and the forced swim test (mood). 3. To evaluate the chemoprevention/antioxidant properties of plant extracts and active compounds. During the previous granting period, we have developed a variety of antioxidant/chemoprevention assays including scavenging of stable free radicals, inhibition of DNA damage induced by the quinone menadione using the comet assay, and induction of quinone reductase. In this aim, extracts of the above mentioned botanical dietary supplements will be fractionated and activity will be determined using the antioxidant/chemoprevention assays. The completion of these aims will determine which specific botanicals have health benefits for relief of menopausal symptoms and determine their mechanism of action.